As an important biomarker, thrombin (TB) is a major player in thrombosis and hemostasis and has attracted increasing attention involving its determination. Herein a universal and ultrasensitive fluorescence biosensor based on a binding-induced 3D-bipedal DNA walker and catalytic hairpin assembly (CHA) strategy has been proposed for cascade signal amplification detection of thrombin. In this study, we designed two proximity probes (foot 1 and foot 2) which include a specific affinity ligand for TB binding and a Pb2+-dependent DNAzyme tail sequence. In the presence of TB, the simultaneous binding of TB to foot 1 (F1) and foot 2 (F2) via TB aptamer (TBA) brings the tail sequences into close proximity and the melting temperature for tail sequences and track DNA is increased, allowing the Pb2+-dependent DNAzyme to cleave the track DNA into two short fragments which have lower affinities for the DNAzyme and, finally, leading to the release of trigger DNA (T-DNA) for subsequent CHA reaction. In the meantime, the dissociated DNA walkers (F1 and F2) explore adjacent unwound track DNA, and the walking procedure is conducted. Unlike the conventional unipedal DNA walkers that anchor foot DNA and track DNA on the same sensing surface, the proposed 3D-bipedal DNA walking machine can not only increase the local concentration of track DNA but can also improve the walking efficiency and expand the range of the walkers to some extent due to the two free feet. Moreover, with the advantages of superior sensitivity and excellent specificity, this biosensing platform exhibits a huge potential in practical application in biomedical research and clinical diagnosis.